Generation of human spinal cord neural stem cells

Abstract

Spinal cord neural stem cells (NSCs) have great potential to reconstitute damaged spinal neural circuitry. In some embodiments, derivation of spinal cord NSCs from human pluripotent stem cells (hPSCs) is described. These spinal cord NSCs can differentiate into a diverse population of spinal cord neurons comprising multiple positions in the dorso-ventral axis, and can be maintained for prolonged time periods. After grafting into injured spinal cords, grafts may be rich with excitatory neurons, extend large numbers of axons over long distances, innervate their target structures, and enable robust corticospinal regeneration. In some embodiments, hPSC-derived spinal cord NSCs enable a broad range of biomedical applications for in vitro disease modeling, and can provide a clinically-translatable cell source for spinal cord “replacement” strategies in several spinal cord disorders.

What is claimed is: 
     
         1 . A method of generating spinal cord neural stem cells comprising, contacting human pluripotent stem cells in a stem cell-appropriate medium with no more than about 4 μM CHIR99021, no more than about 10 μM SB431542, about 100 nM LDN193189, no more than about 100 ng/ml FGF-2, and about no more than about 100 ng/ml FGF-8b, whereby the neural stem cell are induced to have a spinal cord positional identity. 
     
     
         2 . The method of  claim 1 , wherein the concentration of CHIR99021 is about 3 uM. 
     
     
         3 . The method of  claim 1 , wherein the concentration of SB431542 is about 5 μM. 
     
     
         4 . The method of  claim 1 , wherein the concentration of LDN193189 is about 100 nM. 
     
     
         5 . The method of  claim 1 , wherein the concentration of FGF-2 is about 25 ng/ml. 
     
     
         6 . The method of  claim 1 , wherein the concentration of FGF-8b is about 25 ng/ml. 
     
     
         7 . The method of  claim 1 , wherein the culture does not contain DAPT. 
     
     
         8 . The method of  claim 1 , wherein the human pluripotent stem cells are embryonic stem cells. 
     
     
         9 . The method of  claim 1 , wherein the cells are cultured for 7-10 days to produce spinal cord neural stem cells. 
     
     
         10 . The method of  claim 1 , wherein the method further comprises maintaining the spinal cord neural stem cells in medium supplemented with about 1-5 μM CHIR99021, about 1-10 μM SB431542, about 10-50 ng/ml FGF-2, and about 10-50 ng/ml FGF-8b for an additional 30-60 days prior to further use of the cells. 
     
     
         11 . The method of  claim 1 , wherein the spinal cord neural stem cells are karyotypically stable. 
     
     
         12 . The method of  claim 11 , wherein the spinal cord neural stem cells have 0-1 karyotypic abnormalities per preparation. 
     
     
         13 . A spinal cord neural stem cell generated according to the method of  claim 1 . 
     
     
         14 . A method of engrafting a spinal cord neural stem cell into a subject in need thereof comprising implanting the spinal cord neural stem cell of  claim 13  into the subject. 
     
     
         15 . The method of  claim 14 , wherein the subject has a spinal cord injury. 
     
     
         16 . The method of  claim 14 , wherein the subject has a neurodegenerative disease. 
     
     
         17 . The method of  claim 14 , wherein the engrafted spinal cord neural cell survives at least 3-months post engraftment. 
     
     
         18 . The method of  claim 14 , wherein the engrafted spinal cord neural cell undergoes neuronal and glial differentiation in vivo. 
     
     
         19 . A method of generating karyotopically stable spinal cord neural stem cells comprising, contacting human pluripotent stem cells in a stem cell-appropriate medium with no more than about 4 μM CHIR99021, no more than about 10 μM SB431542, about 100 nM LDN193189, no more than about 100 ng/ml FGF-2, and no more than about 100 ng/ml FGF-8b, wherein the spinal cord neural stem cells are karyotypically stable. 
     
     
         20 . The method of  claim 19 , wherein the spinal cord neural stem cells (1) demonstrate increased engraftment, (2) demonstrate increased survival, or (3) undergo neuronal and glial differentiation in vivo.